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BIOLOGY 
LIBRARY 


On  the  Question  of  the  Occur- 
rence of  Creatinine  and 
Creatine  in  Blood 


By 

JEANETTE  ALLEN  BEHRE 

and 
STANLEY  R.  BENEDICT 


Reprinted  from  an  article  based  upon  a  thesis  presented  to 
the  faculty  of  the  Graduate  School  of  Cornell  University  for 
the  degree  of  Doctor  of  Philosophy  by  Jeanette  Allen  Behre. 


. 


The  typewritten  copy  of  the  thesis  upon  which  this  article 
is  based  is  on  file  in  the  Library  of  Cornell  University. 


On  the  Question  of  the  Occurrence  of 
Creatinine  and  Creatine  in  Blood 


A   THESIS 

PRESENTED  TO  THE  FACULTY  OF  THE  GRADUATE  SCHOOL 
OF  CORNELL  UNIVERSITY  FOR  THE  DEGREE  OF 

DOCTOR   OF   PHILOSOPHY 


fcY 

JEANETTE   ALLEN    BEHRE 

i! 


Reprinted  from  JOURNAL  OF  BIOLOGICAL  CHEMISTRY, 
VOL.  LIT,  No.  1,  MAY,  1922. 


BIOLOGY 

LIBRARY 

G 


Reprinted  from  THE  JOURNAL  OF  BIOLOGICAL.  CHEMISTRY 
Vol.  LII,  No.  1,  May,  1922 


STUDIES  IN  CREATINE  AND  CREATININE  METABOLISM. 

IV.     ON  THE  QUESTION  OF  THE  OCCURRENCE  OF  CREATININE 
AND  CREATINE  IN  BLOOD. 

BY  JEANETTE  ALLEN  BEHRE  AND  STANLEY  R.  BENEDICT. 

(From   the   W.  A.  Clark  Special  Research  Fund  and  the  Department  of 
Chemistry,   Cornell   University  Medical  College,  New  York  City.) 

(Received  for  publication,  February  14,1922.) 

The  very  rapid  advances  in  analytical  technique  applied  to 
biological  tissues  and  fluids  made  during  the  past  10  years  have 
resulted  in  a  rather  anomolous  situation.  We  are  constantly 
determining  substances  whose  existence  in  the  tissues  or  fluids 
analyzed  has  never  been  proved,  and  on  the  basis  of  a  single,  non- 
specific color  reaction  reports  are  made  of  the  quantity  of  a  sub- 
stance in  a  given  tissue  or  fluid,  although  none  of  this  substance 
has  ever  been  separated  as  such  from  the  material  analyzed.  The 
modern  color  reactions  are  very  attractive  playthings,  but  the 
facility  with  which  they  can  be  employed  should  not  lead  to  neg- 
lect of  the  more  fundamental  work  of  seeking  definitely  to  prove 
exactly  what  these  color  reactions  may  signify. 

It  is  not  intended,  in  this  connection,  to  decry  the  use  of  modern 
analytical  technique  for  blood  and  tissues — such  studies  should  be 
pursued  as  far  as  they  seem  to  promise  results  of  value  from  any 
angle,  but  we  should  keep  in  mind  the  necessity  of  continued  in- 
vestigation of  the  tissues  and  fluids  of  the  organism  from  the 
qualitative  standpoint. 

Over  a  year  ago  one  of  us  encountered  very  anomolous  results 
during  an  investigation  of  the  question  of  the  probable  interference 
of  creatinine  when  using  the  picrate  method  for  determination  of 
blood  sugar.  These  results,  some  of  which  are  cited  below,  were 
so  at  variance  with  known  reactions  of  creatinine  that  they  ap- 
peared to  demonstrate  that  in  some  bloods  at  any  rate,  creatinine 
could  not  be  present  in  anything  like  the  quantity  indicated  by 
the  colorimetric  method  of  Folin  for  determining  this  substance. 
We  were  therefore  led  to  take  up  a  more  detailed  study  of  this 
question,  and  to  include  a  study  of  the  creatine  content  of  blood. 

11 


507831 


12  Creatinine  and  Creatine  in  Blood 

The  question  of  the  existence  of  creatinine  in  blood  has  already 
been  raised  by  others.  Hunter  and  Campbell  (1)  in  1917  reported 
a  careful  study  of  the  question,  based  chiefly  upon  comparing  the 
rate  of  color  development  of  creatinine  in  alkaline  picrate  solution 
with  color  development  in  blood  filtrates  under  corresponding 
conditions.  Curves  were  constructed  which  showed  the  velocity 
of  color  development  in  this  reaction,  the  amount  of  color  being 
plotted  against  the  time.  With  pure  creatinine  solutions  it  was 
found  that  the  curve  was  typical  and  that  solutions  of  different 
concentrations  show  proportionate  color  production.  Various 
blood  filtrates  were  compared  with  creatinine  solutions,  with  the 
result  that  Hunter  and  Campbell  concluded  that  Folin's  picric 
acid  method  can  be  used  to  determine  the  creatinine  in  the  plasma 
with  accuracy,  but  that  there  is  an  additional  substance  in  whole 
blood  (present  in  the  corpuscles)  which  increases  the  color  of  the 
reaction  due  to  creatinine  by  about  50  per  cent,  while  autoclaved 
filtrates  from  blood  and  plasma  contain  a  still  greater  amount  of 
a  substance,  not  creatinine,  which  contributes  50  to  75  per  cent  of 
the  color  usually  attributed  to  creatinine. 

•  These  authors  did  not  question  the  presence  of  a  certain  amount 
of  creatinine  in  the  blood  but  they  suggested  that  a  considerable 
part  of  the  color  was  probably  not  due  to  creatinine. 

Green wald  and  McGuire  (2)  have*  also  studied  certain  factors 
bearing  on  the  question  of  the  true  creatinine  and  creatine  content 
of  the  blood.  These  authors  did  not  draw  definite  conclusions  as 
to  the  probable  occurrence  of  these  substances  in  blood,  but  a 
reading  of  their  paper  convinces  one  that  they  held  serious  doubts 
as  to  the  true  nature  of  the  "  creatinine"  in  blood.  Their  attempts 
to  isolate  creatinine  from  blood  failed  in  every  instance,  and  their 
final  opinion  is  quite  suggestive  of  suspicion  of  the  whole  subject. 
Thus  they  state: 

"For  an  investigation  of  normal  creatine  and  creatinine  metabolism, 
the  methods  are  probably  not  satisfactory.  Until  it  can  be  shown  that 
the  chromogenic  substance  is  really  creatinine,  investigations  in  this  field 
would  seem  to  be  of  doubtful  value." 

The  available  evidence  that  creatinine  exists  in  blood  may  be 
briefly  summarized  as  follows:  (1)  Creatinine  occurs  in  the  urine, 
hence  it  is  probably  present  in  the  blood  and  (2)  the  rate  of  color 


J.  A.  Behre  and  S.  R.  Benedict  13 

development  in  alkaline  picrate  solutions  due  to  the  blood  com- 
ponent, closely  approximates  that  found  for  pure  creatinine  solu-  • 
tions.     Obviously  neither  of  these  arguments  for  the  presence  of   t 
creatinine  in  blood,  in  the  amounts  indicated  by  present  quantita- 
tive methods  is  at  all  conclusive.     Concerning  the  first  argument 
it  may  be  pointed  out  that  creatinine  may  be  produced  by  the 
kidney,  or  the  kidney  may  be  able  to  concentrate  this  substance 
from  dilutions  in  the  blood  very  much  greater  than  is  commonly 
assumed.     The  point  established  by  Hunter  and  Campbell  also  • 
fails  to  demonstrate  the  existence  of  creatinine  in  blood.     An 
unknown  blood  constituent  might  duplicate  the  rate  of  color  pro- 
duction, and  the  possible  presence  of  catalysts  affecting  the  reac- 
tion in  blood  nitrates,  as  suggested  by  Green  wald  (2),  must  be 
taken  into  consideration.    We  are  therefore  of  the  opinion  that  * 
no  results  so  far  available  offer  definite  evidence  of  the  existence 
of  creatinine  in  blood. 

Our  method  of  studying  the  question  has  developed  along 
several  lines.  We  have  applied  certain  reactions  to  both  blood 
filtrates  and  pure  creatinine  solutions,  and  to  creatinine  added  to 
blood,  and  compared  the  relative  behavior  of  true  creatinine  and 
of  the  blood  creatinine,  both  before  and  after  conversion  of  pos- 
sible creatine  into  creatinine.  We  have  also  made  studies  based 
upon  the  use  of  adsorptive  reagents  which  will  remove  true  crea- 
tinine from  pure  solution  or  from  blood  filtrates. 

In  most  of  the  creatinine  determinations  we  have  employed  the 
original  picric  acid  method  of  Folin  (3),  in  which  the  blood  is  •' 
diluted  to  five  times  its  volume  with  saturated  picric  acid,  and  the 
total  solution  saturated  with  picric  acid.     For  carrying  out  this 
saturation  with  dry  picric  acid  we  have  placed  the  mixtures  in  &f 
shaking  machine  for  from  5  to  10  minutes. 

Our  studies  in  connection  with  this  method  have  shown  that  not 
more  than  4  or  5  mg.  of  creatinine  added  to  100  cc.  of  blood  can  be 
recovered  quantitatively  by  this  technique.  Doubling  the  volume 
of  dilution  permits  the  satisfactory  recovery  of  the  larger  quantities 
of  creatinine.  We  have  also  employed  precipitation  by  means  of  **• 
heat  coagulation,  trichloroacetic  acid,  and  tungstic  acid  (Folin  and 
Wu,  4) ,  followed  by  saturation  of  the  filtrate  (after  exact  neutrali- 
zation in  the  case  of  the  trichloroacetic  acid  precipitation)  with 
picric  acid.  All  of  the  picric  acid  employed  has  at  least  fulfilled 


14  Creatinine  and  Creatine  in  Blood 

the  requirements  of  purity  indicated  by  Folin  and  Doisy  (5)  as 
necessary  for  creatinine  determination  in  blood. 

Results  by  the  various  methods  of  precipitation  indicated  that 
for  normal  bloods  the  various  precipitants  yield  parallel  figures, 
while  for  bloods  high  in  color-reacting  substance  the  Folin- Wu 
precipitation  with  tungstic  acid  usually  yields  much  higher  figures 
than  does  the  picric  acid  precipitation.  Heat  coagulation  filtrates 
show  still  higher  figures  (Table  VI) . 

As  mentioned  earlier  in  this  paper,  our  attention  was  first 
directed  to  the  question  of  the  occurrence  of  creatinine  in  blood 
by  the  results  obtained  by  one  of  us  in  a  study  of  the  possible 
interference  by  creatinine  in  the  picrate  method  for  blood  sugar 
determination.  It  was  found  that  creatinine  added  to  blood 
caused  an  increase  in  the  apparent  blood  sugar  equivalent,  in  terms 
of  glucose,  to  about  three  times  the  quantity  of  creatinine  added, 
provided  that  this  was  more  than  about  3  mg.  It  was  also  found 
that  purified  bone-black,  when  used  in  quantities  of  about  1.5  gm. 
for  the  25  cc.  of  blood  picrate  mixture,  after  a  few  minutes  shaking 
would  remove  up  to  25  mg.  of  creatinine  per  100  cc.  of  blood. 
When  the  bone-black  procedure  was  applied  to  bloods  containing 
high  non-protein  nitrogen  it  was  found  that  while  such  bloods  very 
commonly  showed  a  marked  drop  in  sugar  content  after  treatment 
with  bone-black,  in  many  instances  this  drop  bore  no  relationship 
to  the  creatinine  content  of  the  blood.  Furthermore,  some  bloods 
which  gave  a  high  creatinine  content  by  the  regular  method, 
showed  no  drop  whatever  in  the  sugar  content  when  treated  with 
bone-black.1  A  few  typical  figures  in  this  connection  are  given 
in  Table  I. 

^  An  inspection  of  Table  I  shows  that  there  is  little  relationship 
between  the  fall  in  the  sugar  content  of  the  blood  after  treatment 
with  bone-black  and  the  creatinine  content  of  the  blood.  The 
results  obtained  with  Samples  7,  12,  and  13  render  it  very  im- 
probable, or  indeed  impossible,  that  creatinine  should  have  been 

1  It  may  be  noted  here  that  most  bloods  showing  a  high  non-protein 
nitrogen  content  show  a  marked  drop  in  sugar  content  as  determined  by  the 
Folin-Wu  method  after  treatment  with  bone-black,  and  a  still  greater 
drop  with  Lloyd's  reagent.  The  Folin-Wu  procedure  cannot  be  influenced 
by  creatinine,  or  any  other  known  constituent  of  the  blood  except  glucose. 
It  is  hoped  that  this  work  in  connection  with  blood  sugar  determination 
can  be  reported  upon  in  the  near  future. 


J.  A.  Behre  and  S.  R.  Benedict 


15 


present  in  these  samples  in  the  quantities  indicated  by  the  rec- 
ognized method  for  the  determination  of  this  substance.  Thus 
Sample  12,  with  a  creatinine  content  of  8  mg.  per  100  cc.  which 
should  be  equivalent  to  about  24  mg.  of  glucose  in  the  picrate 
method,  shows  no  appreciable  drop  after  treatment  with  bone- 
black,  which  removes  pure  creatinine. 

TABLE  I. 

The  Effect  of  Bone-Black  Treatment  on  Blood  Sugar  Determinations  in 
Relation  to  Blood  Creatinine. 


Sample  No. 

Sugar  without 
treatment 
with  bone-black 
per  100  cc.  of 
blood. 

Sugar  after 
treatment 
with  bone-black 
per  100  cc.  of 
blood. 

Difference  per 
100  cc.  of  blood. 

Creatinine  per 
100  cc.  of  blood. 

mg. 

mg. 

mg. 

mg. 

1 

96 

88 

8 

1.2 

2 

112 

93 

19 

1.2 

3 

150 

132 

18 

2.0 

4 

280 

220 

60 

6.0 

5 

186 

179 

7 

4.2 

6 

125 

110 

15 

3.1 

7 

116 

112 

4 

5.0 

8* 

119 

101 

18 

1.6 

9 

124 

111 

13 

1.7 

10 

137 

122 

15 

1.4 

11 

108 

81 

27 

1.0 

12 

107 

104 

3 

8.3 

13 

142 

142 

0 

4.6 

14 

117 

118 

1 

1.4 

15 

120 

104 

16 

1.4 

*  Figures  on  Samples  8  to  15  were  obtained  by  Dr.  Gertrude  F.  McCann 
in  the  laboratory  of  Dr.  Frederick  M.  Allen.  We  are  indebted  to  Dr. 
McCann  and  Dr.  Allen  for  permission  to  use  this  material. 

Obviously  such  results  as  are  reported  in  Table  I  cannot  do 
more  than  show  that  any  particular  sample  of  blood  does  not  con- 
tain the  creatinine  ascribed  to  it  by  another  and  more  direct 
method  of  determination*  Nevertheless,  such  results  do  throw 
doubt  upon  the  validity  of  the  general  assumption  that  the  picric 
acid  reaction,  as  applied  for  creatinine,  really  determines  that 
compound  in  blood. 


16  Creatinine  and  Creatine  in  Blood 

Hence  we  were  led  to  study  the  question  further.  A  peculiarity 
of  picric  acid  filtrates  from  blood  which  we  have  repeatedly 
noticed,  and  which  has  probably  impressed  others,  is  that  such 
filtrates  are  very  frequently  definitely  darker  in  color  than  pure 
picric  acid  solutions,  without  addition  of  any  alkali.  Thus  a 
colorimetric  reading  often  showed  such  filtrates  to  have  twice  as 
much  color  as  a  saturated  aqueous  solution  of  picric  acid.  This 
observation  indicated  a  tendency  on  the  part  of  some  .blood  con- 
stituent to  reduce  picric  acid  in  acid  solution.  Thus  we  were  led 

» to  try  the  effect  on  color  development  in  picrate  solution  when 
sodium  carbonate  is  used  as  the  alkali  instead  of  sodium  hydroxide, 
and  to  compare  the  results  thus  obtained  with  those  yielded  by 

*  pure  creatinine  under  similar  conditions.  As  an  arbitrary  stand- 
ard for  color  comparison  in  this  connection  we  employed  a  pure 
•aturated  picric  acid  solution,  adding  0.5  cc.  of  20  per  cent  sodium 
carbonate  to  each  10  cc.  of  solution,  and  making  the  readings 
after  10  minutes.  When  sodium  carbonate  is  added  to  picric 
acid  the  depth  of  color  in  the  solution  increases  slightly.  If 
creatinine  is  present,  in  such  quantities  as  we  assume  in  blood  fil- 
trates, this  increase  in  color  is  slightly  greater,  but  is  scarcely  more 
than  detectable,  and  is  not  definitely  proportional  to  the  creatinine 
present.  In  fact,  increasing  the  creatinine  five  times  yields  a 
scarcely  detectable  increase  in  color  when  carbonate  is  used  as  the 
alkali.  When  carbonate  is  added  to  the  picric  acid  filtrate  from 
blood,  there  is  a  marked  increase  in  color,  and  this  color  is  far 
greater  than  could  possibly  be  due  to  the  creatinine  content  of  the 
blood,  as  indicated  by  the  regular  determination.  In  other  words, 
there  is  in  blood  a  substance  other  than  creatinine  which  reduces 

% 

picric  acid  in  the  presence  of  sodium  carbonate.  It  is  fair  to  assume 
that  this  substance  contributes  to  the  reaction  when  hydroxide 
is  employed  as  the  alkali.  Whether  the  reaction  obtained  with 
hydroxide  is  wholly  due  to  the  same  substance  which  reacts 
(incompletely?)  with  carbonate  cannot  be  answered.  The  in- 
crease of  color  in  the  blood  filtrates  with  carbonate  is  usually 
particularly  great  in  bloods  which  showed  an  abnormally  high 
"creatinine"  content,  but  in  many  other  cases  the  increase  due  to 
carbonate  is  not  proportional  to  that  obtained  with  hydroxide. 
This  may  be  due,  as  it  is  in  the  case  of  creatinine,  to  the  fact  that 
the  reaction  given  by  the  blood  compound  is  very  incomplete 


J.  A.  Behre  and  S.  R.  Benedict  17 

in  presence  of  carbonate.  We  are  inclined  to  believe  that  this 
explanation  is  correct,  and  that  the  "creatinine"  in  blood  differs 
(among  other  particulars)  from  creatinine  in  that  it  reacts  more 
strongly  with  picric  acid  in  presence  of  carbonate.  The  evidence 
in  this  connection  is,  we  admit,  inconclusive  beyond  showing  that 
results  with  hydroxide  cannot  be  assumed  to  represent  the  true 
creatinine  content  of  the  blood. 

More  definite  evidence  that  the  "creatinine"  of  blood  is  not 
creatinine  is  furnished  by  experiments  based  upon  the  fact  that 
creatinine  is  destroyed  by  heating  in  alkaline  solution.  We  have 
found  that  if  solutions  containing  up  to  4.5  mg.  actual  concentra- 
tion of  creatinine  per  100  cc.  (corresponding  to  22.5  mg.  of  crea- 
tinine per  100  cc.  of  blood  where  a  1  to  5  dilution  has  been  em- 
ployed) to  which  have  been  added  2  per  cent  of  sodium  hydroxide, 
are  heated  for  1  hour,  the  creatinine  has  been  completely  destroyed 
so  far  as  its  power  of  yielding  color  in  alkaline  picrate  solution  is 
concerned.  When  filtrates  from  normal  blood  precipitated  by 
tungstic  acid  according  to  Folin  and  Wu  (4)  are  heated  with  alkali 
under  similar  conditions,  the  blood  "  creatinine"  remains  practi-' 
cally  unchanged.  When  similar  filtrates  from  blood  to  which 
creatinine  has  been  added  were  treated  in  a  similar  way,  the  added 
creatinine  was  lost,  while  the  blood  creatinine  was  unchanged. 
We  will  cite  two  examples  in  this  connection.  The  tungstic  acid 
filtrate  from  a  sample  of  beef  blood  showed  a  "  creatinine"  content 
for  the  original  blood  of  1.94  mg.  per  100  cc.  20  cc.  of  this  filtrate 
were  treated  with  5  cc.  of  10  per  cent  sodium  hydroxide,  and  the 
tube  was  heated  in  the  water  bath  for  1  hour.  The  solution  was 
then  neutralized  exactly  with  hydrochloric  acid,  saturated  with 
picric  acid,  and  the  creatinine  determined  as  usual.  The  color 
obtained  corresponded  to  1.82  mg.  of  creatinine  per  100  cc.  of 
blood,  against  the  1.94  mg.  found  without  the  treatment  with 
alkali. 

When  2  mg.  of  creatinine  per  100  cc.  were  added  to  this  same 
blood  (and  recovered  quantitatively  in  the  filtrate),  the  reading 
after  heating  with  the  alkali  showed  2.0  mg.  per  100  cc.  against 
1.9  mg.  in  the  original  blood,  and  4.0  mg.  in  the  blood  with  the 
added  creatinine.  In  another  blood  sample  the  filtrate  showed 
1.96  mg.  of  creatinine  before  heating  with  alkali  and  2.1  after  such 
heating.  Addition  of  creatinine  to  this  same  blood  so  that  the 


18  Creatinine  and  Creatine  in  Blood 

filtrate  showed  a  total  of  8.60  mg.  was  then  made.  After  heating 
with  alkali  the  "creatinine"  content  had  fallen  to  2.50  mg., 
against  1.96  mg.  in  the  original  blood.  In  other  words,  more  than 
6  mg.  of  added  creatinine  per  100  cc.  of  blood  were  destroyed, 
while  the  original  blood  "  creatinine"  was  practically  unaffected. 

Additional  experiments  with  alkali  gave  similar  results,  but  they 
also  led  to  the  discovery  that  glucose  heated  under  similar  condi- 
tions with  alkali  may  give  rise  to  products  which  simulate  crea- 
tinine in  the  picric  acid  reaction.  If  the  glucose  concentration  in 
the  original  blood  be  not  greater  than  0.2  per  cent  there  is  no  inter- 
ference due  to  the  split-products,  but  with  higher  glucose  concen- 
trations the  split-products  may  replace  part  of  the  creatinine  lost 
through  the  action  of  the  alkali.  The  results  cited  above  were 
obtained  with  bloods  of  low  sugar  concentration,  and  thus  furnish 
quite  direct  evidence  that  blood  "  creatinine"  is  not  creatinine. 
Our  finding  that  high  concentrations  of  glucose  would  interfere 
with  the  alkali  study  of  the  creatinine  question  on  many  bloods, 
led  us  to  abandon  this  line  of  work  until  we  had  looked  further 
for  a  method  of  attack  less  open  to  possible  objection.  The  ques- 
tion of  the  occurrence  of  creatinine  in  blood  is  of  such  importance 
that  definite  conclusions  with  regard  to  it  should  not  be  based  upon 
a  line  of  study  open  to  possible  objection  along  any  line. 

Our  next  study  was  concerned  with  the  question  of  the  removal 
of  creatinine,  and  of  the  creatinine-reacting  substance,  from  solu- 
tions by  means  of  kaolin. 

Greenwald  and  McGuire  (2)  have  reported  that  kaolin  removes 
creatinine  quantitatively  from  dilute  solutions,  and  based  a  method 
for  determination  of  creatinine  and  creatine  in  blood  upon  this 
fact.  In  their  study  Greenwald  and  McGuire  employed  blood 
filtrates  obtained  by  heat  coagulation  of  the  blood  proteins  in 
presence  of  dilute  acetic  acid,  and  assumed,  upon  the  basis  of  their 
results  with  pure  creatinine,  that  the  creatinine-reacting  substance 
in  blood  was  also  removed  by  treatment  with  kaolin.  They  ap- 
parently failed  to  make  creatinine  determinations  directly  upon 
filtrates  after  treatment  with  kaolin.  Had  they  not  neglected 
this  point,  Greenwald  and  McGuire  could  hardly  have  escaped 
the  conclusion  that  there  is  no  creatinine  in  blood. 

Using  the  heat  coagulation  filtrate  under  the  conditions  pre- 
scribed by  Greenwald  and  McGuire,  we  have  found  that  true 


J.  A.  Behre  and  S.  R.  Benedict 


19 


creatinine  is  removed  from  pure  solution  or  from  blood  filtrates 
quantitatively  up  to  amounts  corresponding  to  about  4  mg.  of 
creatinine  per  100  cc.  of  original  blood.  With  higher  amounts  of 
creatinine  the  removal  by  kaolin  is  not  quantitative.  Employing 
the  heat  coagulation  filtrates,  the  removal  of  the  creatinine- 
reacting  substance  of  the  blood  by  kaolin  is  irregular  and  uncer- 
tain, even  when  only  small  quantities  of  the  substance  are  present. 
In  Table  II  are  cited  typical  results  in  this  connection  upon  one 
species  of  blood  (beef). 

TABLE  II. 

Showing  Results  of  Kaolin  Extraction  of  Filtrate  Obtained  by  Heat 
Coagulation  of  Beef  Blood,  with  and  without  Addition  of  Creatinine. 


6 
fc 

V 

'ft 

1 

Blood  without  added  creatinine.* 

Blood  with  added  creatinine. 

Before 
extraction 
with 
kaolin. 

After 
extraction 
with 
kaolin. 

Amount 
removed 
by 
kaolin 

Before 
extraction 
with 
kaolin. 

After 
extraction 
with 
kaolin. 

Amount 
of 
added 
creatinine. 

Total 
amount  re- 
moved 
by  kaolin. 

mg. 

mg. 

mg. 

mg. 

mg. 

mg. 

mg. 

1 

2.32 

1.50 

0.82 

3.24 

2.18 

0.92 

1.06 

2 

2.32 

1.50 

0.82 

4.75 

1.72 

2.43 

3.03 

3 

2.14 

1.05 

1.07 

5.04 

2.16 

2.90 

2.88 

4 

2.32 

1.50 

0.82 

6.12 

3.36 

3.80 

2.76 

5 

2.37 

1.05 

1.32 

6.10 

1.94 

3.75 

4.16 

6 

3.06 

2.14 

0.92 

8.05 

2.40 

5.00 

5.65 

7 

3.51 

1.83 

1.68 

8.61 

3.52 

5.10 

5.09 

8 

3.21 

1.87 

1.34 

8.61 

4.02 

5.40 

4.59 

*  All  figures  are  in  terms  of  milligrams  of  creatinine  per  100  cc.  of  blood. 

It  will  be  noted  that  the  quantity  of  the  blood  "  creatinine" 
extracted  by  the  kaolin  would  average  from  about  30  to  50  per 
cent.  The  same  results  hold  true  for  the  blood  of  other  species. 
These  results  may  perhaps  explain  the  very  irregular  creatinine 
values  reported  by  Greenwald  and  McGuire,  using  their  method, 
as  compared  with  figures  obtained  for  the  same  samples  by  the 
Folin  method. 

As  a  result  of  our  work  with  kaolin  upon  the  heat  coagulation 
filtrates  we  soon  gained  the  impression  that  the  amount  of  the 
blood  chromogenic  substance  removed  by  the  kaolin  from  these 
filtrates  depended  upon  variable  factors,  which  could  not  be  well 


20  Creatinine  and  Creatine  in  Blood 

controlled.  Hence  we  set  out  to  find  conditions  yielding  more 
constant  results  in  connection  with  the  use  of  kaolin. 

Experiments  showed  that  in  solutions  completely  freed  from 
protein,  kaolin,2  used  in  a  quantity  of  2  gm.  for  25  cc.  of  solution 
will  remove  creatinine  quantitatively  from  solutions  containing 
up  to  about  0.7  mg.  per  100  cc.  actual  concentration,  which  cor- 
responds to  3.5  mg.  of  creatinine  per  100  cc.  of  blood.  The  mix- 
ture with  kaolin  was  shaken  in  a  shaking  machine  for  about  10 
minutes.  The  solutions  we  employed  were  acid  with  picric,  tri- 
chloroacetic,  or  hydrochloric  acids.  Such  conditions  are  safest  for 
extraction  of  creatinine.  Pure  creatinine  is  removed  by  kaolin 
from  neutral  solution,  but  the  presence  of  even  the  minutest 
amount  of  alkali  prevents  this.  Even  calcium  carbonate  will 
interfere  with  removal  of  creatinine  from  a  solution  by  means  of 
kaolin. 

Employing  kaolin  as  above  indicated  upon  blood  filtrates  ob- 
tained after  a  1  to  5  dilution  and  saturation  with  picric  acid  as  in 
the  Folin  method  for  creatinine  determination,  our  results  appear 
to  show  conclusively  that  normal  beef,  dog,  or  human  bloods  con- 
tain no  creatinine  within  the  limit  of  accuracy  of  the  method  as  we 
employed  it.  Creatinine  in  pure  picric  acid  solution,  or  added  to 
blood,  is  practically  quantitatively  removed  up  to  about  3.5  mg. 
per  100  cc.  of  blood,  while  the  creatinine  chromogenic  substance 
in  the  blood  is  unaffected.  A  hundred  or  more  analyses  have 
been  carried  out  along  this  line.  In  no  instance  has  there  been 
an  appreciable  amount  of  the  chromogenic  substance  of  normal 
blood  removed  by  kaolin,  nor  have  we  ever  failed  to  remove 
added  creatinine  satisfactorily  by  the  use  of  kaolin.  Typical 
results  in  connection  with  this  study  are  given  in  Table  III. 

In  connection  with  the  results  reported  in  Table  III  it  should  be 
remembered  that  creatinine  in  pure  saturated  picric  acid  in  con- 
centrations equal  to  those  reported  for  the  bloods,  is  removed  so 
completely  by  the  kaolin  treatment  that  such  filtrates  show  only 

•We  have  employed  several  different  samples  of  kaolin  with  identical 
results.  Eimer  and  Amend's  "Kaolin,  Acid  Washed"  was  the  one  we  used 
chiefly.  We  have  also  used  Eimer  and  Amend's  "Kaolin"  which  we  our- 
selves washed  with  hydrochloric  acid.  Dr.  Greenwald  kindly  sent  us  a 
sample  of  the  kaolin  employed  by  him.  This~  preparation  gave  similar 
results  to  the  other  products  we  used. 


J.  A.|BehreJandlS.  R.  Benedict 


21 


TABLE  III. 


Showing  Removal  by  Kaolin  from  Picric  Acid  Filtrates  of  Creatinine  Added  to 

Blood,  and  That  the  Chromogenic  Substance  Is  Unaffected  by 

Treatment  with  Kaolin. 


Source  of  blood. 

Blood  without  added 
oreatinine.* 

Blood  with  added  creatinine. 

Before 
extraction 
with 
kaolin. 

After 
extraction 
with 
kaolin. 

Before 
extraction 
with 
kaolin. 

After 
extraction 
with 
kaolin. 

Amount 
of 
creatinine 
added. 

Amount 
removed 
by 
kaolin. 

mg. 

mg. 

mg. 

mg. 

mg. 

mg. 

Beef. 

.86 

1.80 

2.91 

2.01 

1.05 

0.90 

14 

.50 

1.35 

2.40 

1.45 

0.90 

0.95 

tt 

.29 

.33 

2.24 

1.29 

0.95 

0.95 

Dog. 

.27 

.18 

1.92 

1.33 

0.65 

0.59 

Beef. 

.29 

.33 

3.45 

1.50 

2.16 

1.95 

tt 

.86 

.80 

4.45 

2.26 

2.59 

2.19 

« 

.29 

.33 

4.05 

1.56 

2.76 

2.49 

<{ 

.90 

.96 

5.04 

2.16 

3.14 

2.88 

tt 

.50 

.35 

4.70 

1.78 

3.20 

2.92 

It 

.86 

.80 

5.25 

2.00 

3.39 

3.25 

Dog. 

.27 

.18 

4.85 

1.47 

3.58 

3.38 

Beef 

.40 

.18 

6.40 

2.37 

4.00 

4.03 

« 

.29 

.33 

5.30 

1.86 

4.01 

3.44 

Human. 

.84 

.71 

4.55 

2.00 

2.71 

2.55 

« 

3.36 

.60 

6.06 

3.90 

2.70 

2.16 

u 

3.60 

.27 

5.76 

3.81 

2.16 

1.95 

ti 

1.57 

.26 

it 

l.OQ 

.15 

it 

1.23 

.02 

it 

1.96 

2.12 

tt 

1.09 

1.25 

M 

1.44 

1.18 

Beef. 

2.28 

2.28 

u 

2.15 

2.35 

« 

2.34 

2.22 

Dog. 

1.21 

1.23 

« 

1.27 

1.18 

t< 

1.88 

1.82 

*  All  figures  are  in  terms  of  milligrams  of  creatinine  per  100  cc.  of  blood. 

about  0.35  mg.  of  creatinine,  which  is  the  amount  usually  obtained 
with  a  blank  against  a  "0.5  mg.  standard"  creatinine  solution. 
The  figures  reported  in  Table  III  are,  as  stated  above,  typical 


22  Creatinine  and  Creatine  in  Blood 

of  the  results  obtained  in  more  than  a  hundred  analyses.  Plasma 
yielded  results  similar  to  those  obtained  from  whole  blood.  We 
have  included  experiments  showing  maximal  removal  of  the  blood 
"creatinine,"  as  well  as  those  in  which  removal  of  added  creatinine 
was  least  satisfactory.  It  appears  that  the  results  reported  in 
Table  III  furnish  convincing  evidence  that  the  bloods  studied  do 
not  contain  creatinine,  or  at  least  do  not  contain  this  substance  in 
excess  of  a  few  hundredths  of  a  milligram  per  100  cc.  Exceptions 
to  this  statement  might  be  taken,  based  upon  occasional  results 
where  kaolin  treatment  lowers  the  creatinine  value  of  the  blood 
by  a  few  tenths  of  a  milligram  per  100  cc.  But  conversely  it  may 
be  noted  that  instances  also  occur  where  the  chromogenic  value  of 
the  blood  is  increased  by  a  few  tenths  of  a  milligram  after  treatment 
with  kaolin.  The  method  of  study  employed  has  its  limits  of 
accuracy.  The  probable  error  for  a  large  number  of  analyses 
would  not,  we  believe,  exceed  about  0.05  mg.  of  creatinine  per  100 
cc.  Our  results  as  a  whole  indicate  that  the  true  creatinine  con- 
tent of  blood  cannot  exceed  this  figure,  and  we  feel  that  it  must 
probably  be  well  below  it. 

Having  demonstrated  the  absence  of  creatinine  in  detectable 
amounts  from  normal  blood,  it  was  considered  important  to  de- 
termine whether  creatinine  accumulates  in  the  blood  in  conditions 
of  renal  insufficiency  or  of  complete  ablation  of  renal  function. 
Results  here  should  indicate  whether  the  creatinine  in  the  urine  is 
secreted  by  a  process  of  concentration  from  undetectable  traces 
which  occur  in  blood,  or  whether  the  kidney  itself  produces 
creatinine.  In  this  connection  we  have  studied  bloods  obtained 
from  human  cases  where  the  kidney  function  was  impaired,  and 
also  bloods  obtained  from  dogs  42  to  72  hours  after  extirpation  of 
the  kidneys  or  after  ligating  the  ureters. 

The  results  in  connection  with  the  high  bloods  are  not  conclu- 
sive on  the  question  of  the  existence  of  true  creatinine  in  these 
bloods.  Human  bloods  yielding  figures  up  to  about  4  mg.  of 
creatinine  per  100  cc.  show  no  loss  of  the  chromogenic  substance 
after  treatment  with  kaolin.  Higher  bloods  show  a  loss,  following 
treatment  with  kaolin,  amounting  to  from  20  to  50  per  cent,  pro- 
vMing  the  initial  dilution  of  these  bloods  is  great  enough  so  that 
the  actual  concentration  of  reacting  substance  is  about  the  same 
as  in  normal  bloods.  Such  filtrates  may  also  lose  a  considerable 


J.  A.  Behre  and  S.  R.  Benedict  23 

percentage  of  color-yielding  power  (70  to  80  per  cent)  after  heating 
with  alkali,  as  detailed  earlier  in  this  paper.  Very  similar  results 
were  obtained  with  the  dog  bloods  after  ablation  of  the  kidney 
function. 

We  should  be  inclined  to  the  view  that  true  creatinine  accumu- 
lates in  the  blood  after  impairment  of  the  kidney  function,  were 
it  not  for  the  following  considerations: 

After  the  demonstration  that  creatinine  does  not  occur  in  normal 
blood  in  detectable  amounts,  more  definite  evidence  is  needed  to 
prove  its  accumulation  in  abnormal  blood  than  one  or  two  non- 
specific reactions.  Failure  of  removal  by  kaolin  or  of  destruction 
by  alkali  may  well  demonstrate  that  a  compound  is  not  creatinine, 
but  reverse  findings  do  not  demonstrate  that  a  substance  is  crea- 
tinine. We  are  especially  cautious  in  subscribing  to  the  view  that 
creatinine  accumulates  in  the  blood  under  the  special  conditions 
above  cited  because  of  the  failure  of  an  isolation  experiment  to 
demonstrate  the  presence  of  this  compound.  By  the  use  of 
Lloyd's  reagent  a  technique  was  developed  which  permits  isolation 
of  minute  amounts  of  creatinine  from  large  volumes  of  solution. 
So  far  only  a  single  isolation  experiment  has  been  carried  out  in 
this  connection,  upon  blood  obtained  from  a  dog  after  ligation  of 
the  ureters.  The  result  was  wholly  negative  for  the  isolation  of 
creatinine.  Though  the  final  solution  showed  colorimetrically  a 
creatinine  content  of  about  9  mg.  in  5  cc.  of  solution,  creatinine- 
zinc  chloride  failed  to  separate  from  the  solution  after  standing 
2  days.  After  the  addition  of  5  mg.  of  creatinine  to  this  same 
solution  the  added  creatinine  was  recovered  almost  exactly  as  the 
zinc  salt  within  a  period  of  3  hours. 

It  is  this  result  which  makes  us  feel  that  more  data  are  needed 
before  any  conclusion  can  be  drawn  concerning  the  true  creatinine 
content  of  the  "high  creatinine"  bloods.  In  the  near  future  we 
shall  report  further  data  upon  this  problem  together  with  full 
details  of  our  isolation  experiments.  Studies  on  the  muscle  crea- 
tinine are  also  contemplated. 

After  our  investigation  of  the  creatinine  content  of  the  blood  we 
were  led  to  study  the  behavior  of  the  blood  "creatine"  and  to 
attempt  to  find  out  whether  all  or  part  of  this  represents  true 
creatine.  The  study  has  been  complicated  by  the  lack  of  an 
adequate  method  for  conversion  of  creatine  into  creatinine  in  the 


24  Creatinine  and  Creatine  in  Blood 

concentration  found  in  the  blood,  which  can  be  relied  upon  not  to 
yield  decomposition  or  other  products  which  seriously  interfere 
with  the  process. 

A  review  of  the  literature  on  methods  of  creatine  determination 
in  blood  cannot  but  leave  one  with  the  impression  that  each  inves- 
tigator, using  a  new  technique,  and  one  seemingly  accurate,  is 
able  to  get  figures  quite  different  from  those,  obtained  by  any 
previous  method. 

The  original  method  for  conversion  of  creatine  into  creatinine 
in  blood  filtrates  was  proposed  by  Folin  (3)  and  consisted  in  heat- 
ing the  filtrates  with  picric  acid.  Wilson  and  Plass  (6)  and  Hunter 
and  Campbell  (1)  have  raised  the  question  of  the  accuracy  of 
creatine  determination  by  this  procedure.  Folin  and  Wu  (4) 
subsequently  recognized  the  fact  that  such  a  procedure  led  to  high 
results  for  the  "  total  creatinine"  and  suggested  that  traces  of 
hydrogen  sulfide  were  formed  during  such  heating.  Folin  and  Wu, 
however,  did  not  seem  to  give  much  weight  to  their  own  sugges- 
tion, for  their  new  method  for  blood  creatine  included  autoclaving 
blood  filtrates,  and  it  is  not  apparent  why  picric  acid  filtrates 
should  evolve  hydrogen  sulfide  any  more  easily  than  should  any 
other  blood  filtrate. 

Our  experiments  in  connection  with  the  picric  acid  filtrates 
obtained  from  blood  have  shown  that  blood  contains  some  sub- 
stance other  than  creatine,  which  rapidly  reacts  with  picric  acid 
in  hot  solution  to  give  a  product  yielding  color  on  addition  of 
alkali.  After  heating  the  picric  acid  filtrates  from  normal  bloods 
in  the  water  bath  for  1  hour  the  "  creatinine"  value  is  usually 
doubled,  and  after  3  hours  heating  the  value  is  about  trebled.  In 
a  case  of  human  nephritic  blood  we  have  obtained  an  increase  in 
color-yielding  power  equivalent  to  6  mg.  of  creatinine  after  15 
minutes  heating  of  the  picric  acid  filtrate  in  boiling  water.  The 
increases  due  to  such  heating  are  out  of  all  proportion  to  possible 
conversion  of  pure  creatine  into  creatinine  under  identical  condi- 
tions. Similar  results  are  not  obtained  if  other  acids  (acetic, 
trichloroacetic,  hydrochloric)  are  substituted  for  the  picric  acid. 
Hence  the  reaction  is  not  a  change  in  the  blood  constituent  due  to 
heating  with  dilute  acid,  but  is  a  specific  reaction  between  the 
blood  compound  and  picric  acid  in  hot  solution.  Apparently  the 
compound  giving  rise  to  this  reaction  is  not  the  same  as  that  re- 


J.  A.  Behre  and  S.  R.  Benedict  25 

sponsible  for  the  creatine  reaction  of  the  blood  filtrate  when 
heated  with  acid  in  the  absence  of  picric  acid,  for  after  treatment 
which  will  completely  convert  creatine  into  creatinine  there  is 
still  an  increase  if  the  solutions  are  heated  with  picric  acid  prior  to 
addition  of  alkali,  but  the  increase  in  color  is  not  as  great  as  before 
treatment  with  acid,  due  probably  to  the  conversion  of  the  creatine. 
If  filtrates  from  plasma  instead  of  whole  blood  be  heated  after 
saturation  with  picric  acid  the  curve  of  increase  in  color-yield 
after  addition  of  alkali  is  similar  to  that  for  whole  blood,  but  the 
actual  increase  is  only  about  one-half  as  great  (cf.  also  Wilson 
and  Plass,  6).  We  have  no  clue  as  to  the  nature  of  the  substance 
reacting  in  hot  picric  acid  solution  to  yield  the  increased  color. 
Neither  glucose  nor  other  known  constituents  of  the  blood  can 
apparently  be  responsible  for  the  reaction.  The  substance  is 
partially,  but  not  completely,  removed  by  treatment  with  kaolin. 

Although  blood  contains  this  substance  simulating  creatine 
when  heated  with  picric  acid,  it  appears  probable  that  the  creatine 
figures  obtained  for  normal,  and  for  some  pathological  bloods, 
represent  real  creatine.  For  our  creatine  determinations  we  have 
adopted  the  procedure  of  precipitating  the  blood  proteins  by  dilu- 
tion of  the  blood  with  4  volumes  of  5  per  cent  trichloroacetic  acid. 
After  filtration  a  portion  of  the  filtrate  is  treated  with  hydrochloric 
acid  (5  cc.  of  1.0  «N  acid  to  25  cc.  of  filtrate)  and  boiled  down  to  a 
small  volume.  A  little  granulated  or  powdered  metallic  lead  is 
then  added,  and  the  solution  taken  to  dryness  and  heated  on  the 
water  bath  to  expel  the  hydrochloric  acid.3  The  residue  is  dis- 
solved in  water  and  treated  with  sodium  hydroxide,  drop  by  drop, 
until  a  permanent  precipitate  (of  lead  hydroxide)  is  produced. 
The  solution  is  then  made  up  to  a  definite  volume  and  filtered.  A 
portion  of  the  filtrate  is  saturated  with  dry  picric  acid  and  the 
creatinine  determined  as  usual  after  filtration.  The  10  per  cent 
sodium  hydroxide  employed  here  should  contain  10  per  cent  of 
Rochelle  salt. 

This  procedure  gives  a  satisfactory  conversion  of  pure  creatine 
and  is,  we  believe,  reasonably  accurate  for  the  creatine  determina- 
tion in  normal  bloods.  While  satisfactory  for  some  pathological 

1  This  method  is  similar  to  the  one  recommended  by  one  of  us  for  the 
determination  of  creatine  in  urine  (Benedict,  S.  R.,  J.  Biol.  Chem.,  1914, 
xviii,  191.) 


26  Creatinine  and  Creatine  in  Blood 

bloods,  we  are  inclined  to  the  view  that  in  others  no  method  of 
creatine  determination  so  far  developed  can  yield  results  which 
represent  the  true  creatine  content  very  closely. 

When  we  applied  the  procedures  of  heating  with  alkali  and 
treatment  with  kaolin  as  described  earlier  in  this  paper,  to  blood 
filtrates  after  heating  with  acid,  and  compared  the  "  total  creat- 
inine" before  and  after  such  procedures,  we  found  that  for  normal 
bloods  the  color-producing  substance  which  resulted  from  the 
action  of  acid  was  destroyed  by  alkali  and  removed  by  treatment 
with  kaolin  exactly  as  is  pure  creatinine.  Creatine  added  to 
blood  behaved  in  a  similar  manner.  These  findings  hold  for  beef 
and  for  dog  blood.  We  have  not  yet  had  sufficient  normal  human 
bloods  to  warrant  conclusions  concerning  them,  but  we  believe 
that  creatine  actually  exists  in  such  blood. 

The  findings  in  connection  with  the  creatine  content  of  blood  are 
of  interest  also  in  relation  to  the  question  of  the  nature  of  the 
creatinine  chromogenic  substance  in  the  blood.  If  this  latter 
substance  represents  a  loosely  combined  form  of  creatinine,  or  some 
similar  compound,  we  might  expect  that  boiling  with  acid  should 
convert  it  into  true  creatinine.  Yet  after  treatment  to  convert 
creatine  into  creatinine,  it  is  possible  by  means  of  the  action  of 
alkali  or  of  kaolin,  to  separate  practically  quantitatively  the  creat- 
inine derived  from  creatine  from  the  chromogenic  substance 
originally  present  in  the  blood.  Typical  results  in  this  connection 
are  presented  in  Tables  IV  and  V. 

The  results  recorded  in  Tables  IV  and  V  serve  to  show  how  dif- 
ferently the  creatine  of  normal  blood  behaves  after  conversion  to 
creatinine  as  compared  with  the  chromogenic  substance  originally 
present  in  the  blood.  The  remarkably  sharp  separations  effected 
by  means  of  alkali  or  of  kaolin  are  very  striking,  and  serve  to 
further  substantiate  our  earlier  conclusion  that  preformed  crea- 
tinine does  not  exist  in  blood. 

The  view  that  creatine  occurs  in  blood  in  very  appreciable 
amounts  under  certain  conditions  finds  confirmation  in  an  isola- 
tion experiment,  in  which  creatine  was  isolated  as  creatinine-zinc 
chloride.  In  this  experiment  the  blood  obtained  from  a  dog  46 
hours  after  ligation  of  both  ureters  was  employed.  The  total  non- 
protein  nitrogen  of  this  blood  was  186  mg.  per  100  cc.,  and  the 
blood  showed  a  preformed  "creatinine"  content  of  12  mg.,  and  a 


J.  A.  Behre  and  S.  R.  Benedict 


27 


TABLE  IV. 

Showing  the  Removal  by  Kaolin  of  Blood  Creatine  and  of  Creatine  Added 

to  Blood  After  Treatment  for  Conversion  of  Creatine  to  Creatinine, 

and  the  Non-Removal  of  the  Original  "Creatinine"  in  the 

Blood  by  means  of  Kaolin  after  the  Boiling  with  Acid. 


Source  of  sample. 

"Preformed 
creatinine."* 

"Total 
creatinine." 

1 

w 

1 

3 

ofl 

111 

Amount  of  ad- 
ded creatine 
removed. 

Before 
kaolin 
treat- 
ment. 

After 
kaolin 
treat- 
ment 

Before 
kaolin 
treat- 
ment . 

After 
kaolin 
treat- 
ment. 

mg. 

mg. 

mg. 

mg. 

mg. 

mg. 

mg. 

mg. 

Beef. 

2.67 

2.40 

4.35 

2.22 

1.68 

1.68+ 

2.92 

2.72 

" 

2.75 

3.75 

2.10 

0.93 

0.93+ 

3.55 

3.75 

a 

2.22 

1.96 

3.48 

2.04 

1.00 

1.00 

it 

2.22 

1.96 

3.21 

1.70 

1.00 

1.00 

" 

1.21 

1.23 

3.30 

1.59 

2.09 

1.71 

"\rirr 

2.67 

2.40 

4.35 

2.22 

1.68 

1.68 

LJOg 

(ureters  ligated). 

~)f)fT 

6.35 

2.96 

15.30 

2.30 

8.95 

8.95 

L^Ug 

(ureters  ligated). 

4.52 

2.02 

14.30 

1.86 

9.78 

9.78+ 

*  All  figures  refer  to  milligrams  per  100  cc.  of  blood,  in  terms  of  creatinine. 


TABLE  V. 


Showing  the  Destruction  by  Alkali  of  the  Blood  Creatine  and  of  Creatine 

Added  to  Blood,  after  Treatment  for  Its  Conversion  to  Creatinine,  and 

the  Failure  of  Alkali  to  Destroy  the  Original  "Creatinine." 


Blood 

1 

§' 

t 

before  creatine 

•43 

a 

fjj 

conversion.* 

J"2 

9 

8S 

Source  of  sample. 

£ 

0  o 

g 

0  0 

Before 

After 

T3 

•gta 

73 

B 

o>  In 

alkaline 

alkaline 

8 

o  v 
o-o 

1 

TJO^ 

T3^ 

heating. 

heating. 

« 

PQ 

3 

<1 

mg. 

mg. 

mg. 

mg. 

mg. 

mg. 

Beef. 

3.15 

3.15 

2.60 

2.60 

3.4 

2.00 

« 

2.18 

2.00 

2.02 

1.40 

7.7 

5.40 

it 

2.30 

2.30 

2.80 

2.75 

4.58 

4.36 

{( 

2.26 

2.38 

3.09 

2.75 

Dog  with  ligated  ureters. 

4.08 

0.72 

5.62 

5.41 

*  All  figures  refer  to  milligrams  per  100  cc.   of  blood,   in   terms   of 
creatinine. 


28  Creatinine  and  Creatine  in  Blood 

creatine  content  of  16.8  mg.  per  100  cc.  The  preformed  creatinine 
chromogenic  substance  was  practically  completely  removed  by  the 
use  of  Lloyd's  reagent,  while  the  creatine  was  unaffected.  The 
filtrate  was  then  boiled  with  acid  and  the  creatinine  content  of  a 
portion  of  the  solution  determined.  The  main  portion  was  treated 
with  Lloyd's  reagent,  which  removed  the  chromogenic  substance 
practically  quantitatively.  Thus  two  portions  of  Lloyd's  reagent 
were  obtained,  one  containing  the  preformed  chromogenic  sub- 
stance of  the  blood,  the  second  containing  the  chromogenic  sub- 
stance resulting  from  boiling  with  acid.  Both' portions  of  the 
Lloyd's  reagent  were  treated  in  similar  manner  to  liberate  the 
chromogenic  substance,  and  portions  of  the  solutions  thus  obtained 
were  analyzed  colorimetrically  and  each  was  found  to  contain  a 
chromogenic  compound.  The  solutions  were  boiled  down  to  very 
small  volumes  (about  5  cc.)  and  washed  into  small  centrifuge  tubes 
with  the  help  of  alcohol.  The  total  volume  in  each  tube  was  about 
8  cc.;  a  few  drops  of  zinc  chloride  solution  were  added  to  each 
tube,  together  with  a  few  drops  of  a  mixture  of  acetic  acid  and 
sodium  acetate.  Within  half  an  hour  typical  creatinine-zinc  chlo- 
ride crystals  began  to  separate  from  the  tube  containing  creatinine 
derived  from  the  creatine  in  the  blood.  After  standing  over  night 
these  crystals  were  centrifuged.  washed  with  alcohol,  and  dissolved 
in  a  few  drops  of  hydrochloric  acid,  and  the  solution  thus  obtained 
was  diluted  to  a  definite  volume  and  analyzed  for  creatinine  colori- 
metrically. It  was  found  that  10.2  mg.  of  creatinine  had  been 
recovered  as  the  zinc  salt,  against  a  theoretical  value  of  13.6  mg. 
as  determined  in  a  portion  of  the  blood  filtrate  after  hydrolysis. 
As  mentioned  earlier  in  this  paper,  none  of  the  zinc  salt  was  ob- 
tained during  3  days  from  the  tube  containing  the  preformed 
chromogenic  substance  in  the  blood,  though  addition  of  a  small 
amount  of  pure  creatinine  to  the  final  solution  promptly  resulted 
in  the  separation  of  typical  crystals  of  the  zinc  compound.  Full 
details  of  these  isolation  experiments  will  be  reported  later. 

In  regard  to  the  question  of  the  occurrence  of  creatine  in  blood 
of  patients  with  renal  insufficiency  we  should  state  that  our  results 
have  been  so  contradictory  that  we  are  led  to  believe  that  some  of 
these  bloods  contain  large  quantities  of  one  or  more  interfering 
substances  in  the  creatine  determination,  while  in  others  the  crea- 
tine figures  may  be  fairly  exact.  Some  nephritic  bloods  show 


J.  A.  Behre  and  S.  R.  Benedict  29 

removal  of  the  creatinine  derived  from  creatine  by  means  of  kaolin, 
while  others  behave  very  differently.  In  one  case  of  bichloride 
poisoning  with  a  very  high  apparent  creatine  we  failed  to  effect 
practically  any  removal  of  the  chromogenic  substance  resulting 
from  the  action  of  acid,  by  means  of  kaolin  treatment.  Our  results 
indicate  that  cases  may  fall  into  definite  groups  in  regard  to  the 
true  creatine  content  and  the  study  of  the  question  is  being 
continued. 

DISCUSSION. 

Our  finding  that  creatinine  does  not  exist  in  blood  in  detectable 
quantities  need  not,  of  course,  raise  any  question  as  to  the  value  of 
the  determination  of  the  chromogenic  substance  for  clinical  or 
other  purposes.  In  connection  with  such  determination  our  work 
has  brought  out  some  points  which  may  be  of  interest.  In  the 
first  place  there  is  the  question  of  complete  saturation  of  the  solu- 
tions with  picric  acid  prior  to  the  creatinine  determination.  This 
point  has  been  emphasized  by  Greenwald  and  McGuire,  but  we 
believe  that  it  has  not  received  sufficient  recognition.  For  any 
method  of  creatinine  determination  in  blood,  except  the  procedure 
advocated  by  Folin  and  Wu,  it  is  necessary  to  saturate  a  solution 
or  mixture  with  solid  picric  acid.  The  Folin- Wu  procedure,  while 
perhaps  theoretically  preferable  to  the  earlier  methods  from  cer- 
tain standpoints,  yields  such  weak  colors  that  we  question  the 
general  usefulness  of  the  method.  Few  analysts  can  read  such 
colors  with  even  approximate  accuracy  unless  the  bloods  are  very 
high  in  chromogenic  substance.  We  therefore  believe  that  satura- 
tion with  picric  acid  is  preferable  in  all  ordinary  work.  Such 
saturation  may,  of  course,  be  applied  to  any  filtrates  (such  as  the 
Folin- Wu  tungstic  acid  filtrate)  as  well  as  to  the  original  blood. 

In  any  case,  we  believe  it  essential  that  laboratories  where 
attempted  saturation  with  picric  acid  is  a  routine  procedure  should 
be  equipped  with  shaking  machines  (a  rotary  type  is  satisfactory), 
and  that  all  solutions  to  be  saturated  with  picric  acid  should  be 
placed  in  such  machines  for  from  5  to  10  minutes,  prior  to  filtra- 
tion. When  numerous  bloods  are  handled  it  is  probable  that  in 
no  other  way  can  even  approximate  saturation  with  picric  acid  be 
secured.  Where  Myers'  method,  which  involves  preliminary  dilu- 
tion of  the  blood  with  distilled  water,  is  employed,  especial  care 


30  Creatinine  and  Creatine  in  Blood 

has  to  be  taken  in  regard  to  saturation  with  the  picric  acid.  In 
this  method  the  preliminary  stirring  as  recommended  by  Myers 
should  be  followed  by  from  5  to  10  minutes  in  a  shaking  machine. 
While  it  is  possible  to  saturate  a  blood  as  recommended  by  Myers, 
our  experience  shows  that  this  is  rarely  accomplished  in  practice, 
and  that  where  many  bloods  are  handled  simultaneously,  it  is 
very  difficult  to  secure  saturation  by  means  of  stirring.  The 
necessity  for  full  saturation  is  clearly  brought  out  by  Greenwald 
and  McGuire. 

A  second  point  of  interest  in  connection  with  the  technique  for 
the  chromogenic  substance  in  blood  is  in  connection  with  the  pro- 
tein precipitant  employed  and  the  amount  of  dilution  at  the  time 
of  the  protein  precipitation.  As  noted  earlier  in  this  paper,  the 
original  method  of  Folin,  employing  a  1  to  5  dilution  with  saturated 
picric  acid  when  applied  to  bloods  with  high  "creatinine"  values, 
results  in  a  loss  of  a  very  considerable  quantity  of  the  chromogenic 
substance.  This  loss  may  amount  to  5  mg.  or  more,  per  100  cc. 
The  tungstic  acid  precipitation,  carried  out  at  a  1  to  10  dilution  as 
proposed  by  Folin  and  Wu  gives  a  much  better  recovery  of  the 
blood  chromogenic  substance  in  the  abnormal  bloods  than  does 
the  picric  acid  method  at  the  dilution  of  1  to  5.  The  figures  ob- 
tained with  this  filtrate  agree  closely  with  those  given  by  a  precipi- 
tation with  trichloroacetic  acid  in  a  1  to  5  dilution.  Figures  by 
these  methods  are  usually  appreciably  lower  than  where  heat 
coagulation  is  employed  in  a  1  to  5  dilution.  The  difference,  how- 
ever, is  not  great,  and  on  account  of  convenience  we  should  at 
present  recommend  use  of  the  Folin- Wu  precipitation  with  tung- 
stic acid,  followed  by  saturation  of  a  portion  of  the  filtrate  with 
dry  picric  acid  in  a  shaking  machine  for  from  5  to  10  minutes. 
After  filtering  from  the  excess  of  picric  acid  the  chromogenic  sub- 
stance is  determined  as  in  the  original  Folin  method,  using  stan- 
dard creatinine  solutions  in  saturated  picric  acid.  Table  VI  shows 
some  comparative  figures  for  the  chromogenic  substance  in  dog 
bloods  rich  in  this  substance  where  different  protein  precipitants 
were  employed. 

The  question  naturally  arises  as  to  the  possible  bearing  upon 
theories  of  creatine  and  creatinine  metabolism  of  the  findings 
reported  in  this  paper.  While  we  feel  that  the  present  data  do  not 
warrant  detailed  discussion,  there  are  certain  points  brought  up 
by  the  present  work  which  may  be  briefly  considered. 


J.  A.  Behre  and  S.  R.  Benedict 


31 


If  creatinine  cannot  be  demonstrated  in  the  blood,  there  are  two 
possible  sources  for  its  presence  in  the  urine.  It  may  be  present 
in  the  blood  in  traces,  and  the  kidney  may  be  able  to  concentrate 
creatinine  from  this  exceedingly  dilute  solution.  If  this  view  is 
correct,  we  should,  as  pointed  out  earlier  in  this  paper,  be  able  to 
demonstrate  creatinine  in  blood  after  impairment  or  ablation  of 
the  kidney  function.  No  demonstration  short  of  isolation  will 
suffice  in  this  connection.  In  view  of  the  failure  of  isolation  experi- 
ments reported  so  far,  it  would  seem  of  interest  to  discuss  the 
possible  origin  of  the  urinary  creatinine  if  creatinine  does  not  occur 

TABLE  VI. 

Apparent  Creatinine  by  the  Different  Methods  for  Blood  of  Dogs  with  Ureters 

Ligated. 


Source    f  blood. 

Hours  between 
operation 
and  withdrawal 
of  blood. 

Apparent  creatinine  in  mg.  per  100  cc.  of  blood. 

Picric  acid 
method 
1:5  dilution. 

Sodium 
tungatate 
precipitation 
1:10  dilution. 

Trichloro- 
acetic  acid 
precipitation 
1:10  dilution. 

Heat 
coagulation 
method 
1:5  dilution. 

hrs. 

Dogl 
"     2 

48 
72 

4.85 

6.55 

6.96 

8.58 

"    3 

42 

7.35 

11.6 

11.1 

12.0 

"    4 

46 

6.37 

8.10 

9.04 

10.3 

"     5 

46 

11.10 

12.24 

12.24 

14.52 

in  the  blood.  Taking  this  latter  view,  we  must  assume  that  the 
kidney  itself  produces  creatinine  from  some  precursor  substance  in 
the  blood.  The  most  probable  precursor,  from  the  chemical  stand- 
point, would,  of  course,  be  creatine.  It  is  in  line  with  this  hypoth- 
esis that  we  find  real  evidence  for  the  existence  of  creatine  in 
blood,  and  it  is  reasonably  certain  that  under  normal  conditions 
the  creatine  content  of  the  blood  is  very  appreciable.  What  then 
is  the  fate  of  this  creatine?  Is  it  a  waste  product,  or  is  it  to  be 
built  up  into  body  tissue?  The  positive  result  of  our  isolation 
experiment  on  the  creatine  of  the  blood  of  a  dog  after  ablation  of 
the  excretory  function  of  the  kidney,  where  we  were  able  to  demon- 
strate an  accumulation  of  creatine  in  this  blood  to  an  extent  ex- 
ceeding 13  mg.  per  100  cc.,  would  seem  to  show  that  the  creatine 
of  the  blood  is  a  waste  product,  to  be  eliminated  by  the  kidney. 


32  Creatinine  and  Creatine  in  Blood 

All  five  of  our  dogs  in  which  kidney  function  was  ablated  showed 
high  creatine  figures  as  follows:  8.9,  8.1,  9.3,  18.5,  and  16.8  mg. 
per  100  cc.  This  creatine  behaves  as  true  creatine,  and  was  iso- 
lated (as  creatinine)  in  the  only  attempt  we  have  made.  Hence  we 
may  conclude  that  creatine  accumulates  in  the  blood  when  the 
excretory  function  of  the  kidney  is  defective,  and  we  may  further 
conclude  that  creatine  in  the  blood  is  a  waste  product.  Creatine  is 
not  normally  eliminated  as  such  in  the  urine,  hence  it  would  seem 
that  the  kidney  must  eliminate  creatine  as  creatinine  or  as  some 
other  product — perhaps  urea.  An  alternative  view  that  there  is 
some  organ  which  cannot  act  in  destroying  creatine  except  when  the 
kidney  is  performing  its  excretory  function  would  seem  too  extreme 
a  view  to  deserve  consideration.  It  would  seem  then  that  the 
kidney  normally  eliminates  creatine  as  creatinine  or  as  some  other 
product.  Elimination  as  creatinine  seems  far  more  probable. 
There  are,  of  course,  difficulties  in  the  way  of  accepting  this  view. 
The  first  which  suggests  itself  is  the  fact  that  ingested  creatine 
does  not  appear  in  the  urine  as  creatinine  except  in  traces.  Most 
observers  except  Folin,  are  agreed  that  administration  of  creatine 
is  followed  by  an  appreciable,  though  very  slight  increase  in  the 
urinary  creatinine.  The  observations  of  Folin  and  Denis  (7) 
would  explain  how  it  would  be  possible  for  ingested  creatine  to 
fail  to  be  eliminated  in  considerable  amounts  either  as  creatine  or 
creatinine,  even  though  normally  creatine  is  the  source  of  the 
urinary  creatinine.  These  investigators  have  shown  that  mus- 
cular tissue  appears  to  have  a  marked  affinity  for  creatine,  and 
that  this  substance  rapidly  disappears  from  the  blood  and  is 
found  increased  in  the  muscles.  Assuming  even  an  equal  affinity 
for  creatine  between  muscular  and  kidney  tissue,  we  can  readily 
see  why,  on  account  of  the  relative  bulk  of  the  muscular  tissue, 
nearly  all  of  the  creatine  ingested  could  get  into  the  muscles  as 
creatine,  instead  of  into  the  urine  as  creatinine,  even  though 
normally  the  kidney  eliminates  the  creatine  as  creatinine. 
Furthermore,  it  is  quite  possible  that  the  formation  of  creatinine 
in  the  kidney  represents  some  definite  form  of  kidney  metabolism 
associated  with  a  portion  of  its  excretory  function.  Thus  crea- 
tinine might  even  represent  a  measure  of  a  special  type  of  kidney 
metabolism.  It  is  also  possible  that  the  creatine  in  the  circulation 
differs  from  ordinary  creatine,  and  is  in  a  combination  which  cao 
readily  be  converted  into  creatinine. 


J.  A.  Behre  and  S.  R.  Benedict  33 

The  question  of  the  occurrence  of  creatine  in  the  urine  need  not 
be  considered  until  isolation  experiments  have  shown  whether  the 
so  called  creatine  in  urine  is  really  that  compound.  We  are  study- 
ing the  question  by  means  of  a  method  which  we  feel  will  demon- 
strate whether  creatine  occurs  in  urine. 

In  the  meantime  we  feel  that  any  theory  of  creatine  and  crea- 
tinine  metabolism  must  take  into  account  the  fact  that  creatine 
circulating  in  the  blood  appears  to  be  essentially  a  waste  product. 
The  view  above  proposed  attempts  to  do  this,  and  is  the  only 
view  so  far  put  forward  which  takes  account  of  the  increased  blood 
creatine  after  impaired  kidney  function. 

BIBLIOGRAPHY. 

1.  Hunter,  A.,  and  Campbell,  W.  R.,  /.  Biol.  Chem.,  1917,  xxxii,  195. 

2.  Greenwald,  I.,  and  McGuire,  G.,  /.  Biol.  Chem.,  1918,  xxxiv,  103. 

3.  Folin,  O.,  J.  Biol.  Chem.,  1914,  xvii,  475. 

4.  Folin,  O.,  and  Wu,  H.,  J.  Biol.  Chem.,  1919,  xxxviii,  81. 

5.  Folin,  O.,  and  Doisy,  E.  A.,  J.  Biol.  Chem.,  1916-17,  xxviii,  349. 

6.  Wilson,  D.  W.,  and  Plass,  E.  D.,  J.  Biol.  Chem.,  1917,  xxix,  413. 

7.  Folin,  O.,  and  Denis,  W.,  J.  Biol.  Chem.,  1914,  xvii,  493. 


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